Lipid peroxidation (LPO) processes are closely associated with the initiation and early progression stages of atherosclerosis. The antioxidant activity of vitamin C contributes to the termination of radical-mediated LPO processes. However, ascorbic acid also promotes the formation of cytotoxic and potentially atherogenic LPO products via one-electron reduction of lipid hydroperoxides. Thus, the overall role of vitamin C in radical- mediated lipid peroxidation processes remains to be established. The objective for this application is to determine the role of vitamin C in lipid peroxidation processes and how this role contributes to the overall response of vascular endothelial cells and vascular smooth muscle cells to oxidative stress. The central hypothesis to be evaluated is that vitamin C eliminates harmful LPO products, such as 4-hydroxy-2-nonenal (HNE), through ascorbylation (conjugate formation), resulting in an overall anti-atherogenic effect on the vascular wall. We further hypothesize that ascorbylation leads to enhanced plasma levels of these vitamin C conjugates after an acute oxidative stress insult in healthy individuals, whereas ascorbylation is compromised under conditions of chronic oxidative stress, such as smoking and coronary heart disease. Our three Specific Aims are: (1) Identify the biological source of ascorbylated HNE and characterize its disposition, metabolism and excretion, (2) Characterize the atherogenic responses elicited by oxidized lipids and their ascorbyl conjugates in human aortic endothelial cells (HAECs) and in vascular smooth muscle cells (VSMCs), and (3) Determine the relationship between ascorbylated LPO product levels and oxidative stress in smokers, with and without vitamin C supplementation, and in patients with angiographically confirmed coronary artery disease. The research proposed in this application is significant, because elimination of electrophilic LPO products through ascorbylation will have a profound impact on the current understanding of diseases that are exacerbated by lipid peroxidation processes, such as atherosclerosis. This project contributes to our long- term goal to determine the role of lipid peroxidation in inflammatory and age-related disease and how its effects can be modulated by therapeutic or dietary intervention.